Characteristics of Chryseobacterium bacteremia, associated risk factors and their antibiotic susceptibility pattern at a university hospital: a descriptive, retrospective study

Introduction. Chryseobacterium species are emerging bacteria capable of causing nosocomial infections in immunocompromised patients or patients with indwelling medical devices. Hypothesis/ Gap statement. Information about the incidence of Chryseobacterium bacteremia from worldwide literature is limited. Aim. We aimed to recognize the clinical characteristics, frequency of distribution of different Chryseobacterium species isolates, and their antimicrobial susceptibility profile from bloodstream infections. Methods. We performed a retrospective cohort study to identify all isolates of Chryseobacterium species from bloodstream infection from January 2018 to November 2022 at a university hospital in North India. Results. We identified 42 non-duplicate isolates of Chryseobacterium species from bloodstream infection in the duration of our study. Mean age of the patients was 48.35±16.63 years. Men (22/42, 52.2 %) were more commonly affected in comparison to women (20/42, 47.6 %) but the difference was not significant. The most common species identified was C. indologenes (40/42, 95.24 %) followed by C. gleum (2/42, 4.76 %). The co-morbidities commonly encountered in our study were chronic kidney disease (21/42, 50.0 %) followed by diabetes mellitus (12/42, 28.6 %) and chronic obstructive pulmonary disease (8/42, 19.05 %). All patients had intravenous access to medications or fluid management via a central or peripheral line and mechanical ventilation was observed in 39 (39/42, 92.86 %) patients. All the isolates were susceptible to minocycline (100 %), followed by doxycycline (97.6 %) and trimethoprim-sulfamethoxazole (95.2 %). Conclusion. Chryseobacterium species are capable of causing pneumonia, bacteremia and urinary tract infection in immunocompromised patients. Early diagnosis and prompt treatment with appropriate antibiotics can prevent progression to septicemia.


INTRODUCTION
Chryseobacterium spp.are ubiquitous and are found mainly in water and soil [1].In the hospital environment, these bacteria colonize wet and humid surfaces like water tanks, sinks, and washbasins acting as a source of infection [2].Infections due to Chryseobacterium spp.are usually nosocomial and patients acquire infection through colonized medical devices coming in contact with fluids, e.g.respirators, humidifiers, syringes, and contaminated surgically implanted prosthetic devices [3][4][5].Chryseobacterium spp.rarely causes infection in the immunocompetent host.Among immunocompromised patients, Chryseobacterium spp.have been implicated in patients of all age groups.These have been isolated from cases of meningitis, pneumonia, catheter-related bloodstream infections, endocarditis, biliary tract infections, ocular infections, skin and soft tissue infections, peritonitis, urinary tract infections, and surgical wound infection [6][7][8][9].
Chryseobacterium spp.belong to the family Flavobacteriaceae and was previously known as Flavobacterium CDC group IIb.It was first isolated as an opportunistic pathogen from the respiratory secretions of a patient suffering from ventilator-associated pneumonia (VAP) [10].The commonly isolated species from clinical samples include C. odoratum, C. meningosepticum, C. breve, C. multivorum and C. gleum, and C. indologenes.Only C. indologenes, C. gleum, and C. hominis are known to cause infection in humans.Chryseobacterium spp.are Gram-negative, non-lactose fermenting, aerobic, non-motile bacilli producing oxidase and catalase enzymes.This bacterium also produces a distinct yellow-to-orange pigment on blood agar [11].
These bacteria can produce biofilms on foreign medical devices like catheters, and prosthetic implants, and protease production may be the virulence factor involved in the pathogenesis [12].
Nowadays with the increased incidence of carbapenem-resistant and extended-spectrum beta lactamase pathogens like Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae has led to indiscriminate use of drugs of last resort like tigecycline and colistin.This indiscriminate use of antimicrobials of last resort has led to Chryseobacterium species emerging as a significant problem in the critical healthcare setting [13,14].
Intrinsic resistance to first, second, and third generation cephalosporins and carbapenems by virtue of production of molecular class A β-lactamase bla CIA and class B metallo-betalactamase bla IND is observed in Chryseobacterium indologenes [15,16].These exhibit resistance to other classes of antibiotics also which include aminoglycosides, chloramphenicol, clindamycin, tetracyclines, macrolide and teicoplanin, while piperacillin-tazobactam, minocycline, ceftazidime, trimethoprim-sulfamethoxazole, quinolones and rifampicin usually remain effective against this microorganism [2].
Data on antimicrobial susceptibility of Chryseobacterium spp.remains limited owing to its rare isolation from clinical specimens.No specific guidelines for antimicrobial susceptibility testing from CLSI (Clinical Laboratory Standards Institute) or EUCAST (European Committee for Antimicrobial Susceptibility Testing) have been defined for the genus Chryseobacterium spp.
There is limited data available on infections due to Chryseobacterium spp.and their antimicrobial susceptibility profile from across the world.Hence, we conducted this study to recognize the clinical characteristics, risk factors and drug susceptibility pattern of various Chryseobacterium spp.isolated from blood culture samples.

Study design and study population
This is a retrospective observational study where automated blood cultures that grew Chryseobacterium spp.(while no isolate with the identification of Flavobacterium genus was isolated in the study) were assessed for patient characteristics and antibiotic susceptibility patterns from January 2018 to November 2022 in the microbiology laboratory of a university hospital.The study was approved by the Institutional Ethics Committee (2021-48-EMP-EXP dated 29 November 2021).Informed consent was waived given our study is retrospective.

Data collection
A set of automated BACTEC blood culture bottles (Becton Dickinson Diagnostic Instrument Systems, Sparks, MD) were received in the Bacteriology section of the Department of Microbiology and underwent standard microbiological processing like culture followed by microscopy and bacterial isolation.Further, the blood cultures exhibiting suspected growth of Chryseobacterium spp.were identified by a phenotypic method involving the addition of a drop of 10 % potassium hydroxide (KOH) over the colonies leading to change of colour from yellow to red indicating the presence of flexirubin pigment [17].The identification was confirmed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (BioMérieux, United States) in this study.Laboratory data of all the patients was retrospectively collected over the period of 5 years from the laboratory registers and clinical data was retrieved from the hospital information system (HIS) and patient files.

Inclusion criteria
A true pathogen was defined depending on the time of positivity of blood culture and clinical and laboratory indexes in the study.Positive blood culture from samples collected before 48 h of admission were labelled as community-acquired bloodstream infection (BSI) while the positive blood culture collected after 48 h of admission were labelled as nosocomial in origin.

Exclusion criteria
Isolates collected only on a single positive culture without any significant clinical parameters were considered a contaminant and excluded from the study.

Treatment administered
We considered the use of appropriate antibiotics even if one drug from the panel was used in treatment of the patient.

Statistical analysis
Categorical variables were described using percentages and a Chi-square test was applied.Univariate analysis of the risk factors in all patients included in our study was assessed using Kaplan-Meier survival analysis.All statistical analyses were performed using SPSS statistical software (IBM SPSS version 20, Armonk, N.Y.).

RESULTS
In our study, a total of 42 isolates Chryseobacterium indologenes, n=40 (40/42, 95.24 %); and Chryseobacterium gleum, n=2 (2/42, 4.76 %) were identified from bloodstream infections during the course of the 5 year study period.Fig. 1 represents the prevalence of these 42 cases of Chryseobacterium bacteremia by year during the 5 year study period.All the isolates identified in this study were subjected to identification by use of Matrix assisted laser desorption/ ionization-time of flight mass spectrometry (MALDI-TOF MS) and all 42 isolates were included in the study after confirmatory identification using MALDI-TOF MS with a confidence level of 99.9 %.Mean age of the patients in our study cohort was 48.35±16.63years.Men (22/42, 52.2 %) were more commonly affected than women (20/42, 47.6 %) but this difference was not significant.The majority of these patients were admitted to critical care medicine 66.6 % (28/42) followed by anaesthesia 19 % (8/42), emergency 7.1 % (3/42) and other departments including nephrology, cardiology and pulmonary medicine.Forty (40/42, 95.24 %) cases of bacteremia were nosocomially acquired while only two (2/42, 4.76 %) cases were community-acquired.Acute or chronic kidney disease was observed in nine (9/42, 21.43 %) patients followed by seven (7/42, 16.67 %) patients with acute necrotizing pancreatitis and five (5/42, 11.90 %) patients with severe dengue.Other underlying comorbidities include gastrointestinal diseases like intestinal obstruction, Gall bladder perforation following cholecystectomy, and angiodysplasia of the colon in three (3/42, 7.14 %) patients and cardiovascular diseases like valve regurgitation, myocarditis, infective endocarditis, and coronary artery disease in four (4/42, 9.52 %) patients.Indwelling devices were identified as significant predisposing factors attributing to Chryseobacterium bacteremia.All the patients (42/42, 100 %) admitted to our tertiary care centre were managed on intravenous access for medication and fluid management using central or peripheral line and 73.81 % (31/42, 73.81 %) were managed on mechanical ventilation (Table 1).
Co-infections with other bacterial and fungal agents were observed in 30 (30/42, 71.43 %) patients.The co-infection most commonly encountered was with Candida spp.(10/30, 33.33 %) followed by five (5/30, 16.67 %) cases of A. baumannii, five (5/30, 16.67 %) cases of Klebsiella pneumoniae, four (4/30, 13.33 %) cases of Pseudomonas aeruginosa and six (6/30, 20.0 %) cases of Escherichia coli.The mode of administration of all antibiotics to the patients included in this study was by intravenous route to achieve adequate antibiotic concentration in the bloodstream and counteract the life-threatening bacteremia.Keeping in mind the immunocompromised state of the patients in this study it was the most feasible method of drug administration to improve patient outcome.
As observed in the electronic hospital records, the patients who survived were later shifted onto oral antibiotics on discharge.
The overall mortality rate recorded in our study was 54.76 % (23/42).We also compared the demographic and clinical characteristics with the survival in patients with Chryseobacterium bacteremia (Table 1).The underlying comorbidities significantly associated with death in our patient cohort were acute necrotizing pancreatitis, hypertension and diabetes mellitus.The presence of indwelling devices like central venous catheter (P=0.003) in 33 (33/42, 78.57%) patients and mechanical ventilation (P<0.001) in 31 (31/42, 73.81 %) patients were statistically significant risk factors responsible for mortality.Procalcitonin (P<0.001) and total leucocyte counts (P=0.007) were used as a significant parameter for diagnosing septicemia, and higher values of procalcitonin and total leucocyte count were recorded in patients who died.An increased duration of hospitalization was significantly associated with increased deaths among our patient cohort.Cases of nosocomially acquired bacteremia were more commonly associated with death.Appropriate administration of antibiotics in patients who survived was statistically significant in comparison to those who succumbed to Chryseobacterium bacteremia.Only twelve (12/42, 28.57%) patients of Chryseobacterium bacteremia showed antibiotic susceptibility to carbapenems, third-generation cephalosporins and extended spectrum beta-lactams that were being administered empirically, which could be attributed to the rampant use of carbapenems and other drugs for empirical treatment in the hospital, rendering them ineffective and increased association with indwelling devices, we recorded a high rate of mortality in our study cohort.

DISCUSSION
Chryseobacterium infections have been perceived in the extremes of age (premature infants and elderly), immunocompromised patients with chronic obstructive pulmonary disease, diabetes mellitus, chronic kidney disease, cardiovascular disease, malignancies and patients on broad-spectrum antibiotics [18].In this study, common co-morbidities encountered were kidney diseases (22/42, 52.38 %) followed by diabetes mellitus (12/42, 28.57%) and respiratory diseases (10/42, 23.81 %).Chryseobacterium spp.are capable of inflicting a range of infections like bacteremia, pneumonia, urinary tract infection, and biliary tract infection.The repeated isolation of Chryseobacterium spp.from blood specimens was observed in all cases included in this study, signifying its clinical relevance as a bloodstream pathogen which corroborates the results of studies by Chen et al. [12] and Yadav et al. [19].The predisposing risk factors are long-term indwelling devices like endotracheal tubes [20], central and peripheral lines [21] and urinary catheter [22], which was also experienced in this study given an statistically significant number of deaths in comparison to patients who survived with concomitant presence of Chryseobacterium bacteremia and indwelling catheters especially central venous catheters (33/42, 78.57%) and / or mechanical ventilation (31/42, 73.81 %).
The majority of patients were hospitalized in the critical care unit with concomitant presence of indwelling catheters.All patients (42/42, 100 %) in our study cohort had a central and / or peripheral line for intravenous access and 31 (31/42, 73.81 %) were managed for respiratory distress on mechanical ventilator.Thirty-two (32/42, 76.19 %) cases of nosocomially acquired bacteremia and ten (10/42, 23.81 %) cases of community-acquired bacteremia were identified.This nosocomial acquisition of Chryseobacterium bacteremia can be attributed to its property to colonize and form biofilms on humid medical devices like a humidifier, respirators, contaminated catheters and surgical instruments and spread in a hospital setting through contaminated medical devices [18].The above finding was consolidated by a study from North India that reported a history of indwelling medical devices in 55.56 % (10/18, 55.56 %) of their patients [19,23,24].[18].
Chryseobacterium spp.are usually susceptible to minocycline, trimethoprim-sulfamethoxazole, quinolones, and rifampicin [22].All isolates were susceptible to minocycline (100 %), while the isolates were found to be 97.6 and 95.2% susceptible to doxycycline and trimethoprim-sulfamethoxazole, respectively.However, the susceptibility of levofloxacin, piperacillin-tazobactam, cefoperazonesulbactam, ceftazidime, amikacin, ticarcillin-clavulanic acid were 42.8, 30.9, 26.1, 11.9, 9.5 and 9.5% respectively.Chryseobacterium spp.showed 95.3 % resistance to carbapenem.Zhang et al. determined the AST of Chryseobacterium spp.using broth microdilution (BMD) and demonstrated that minocycline (98.5 %) and trimethoprim-sulfamethoxazole (97.8 %) were more potent antimicrobial agents which corresponds to the antibiotic susceptibility pattern observed in our study [22].In a study conducted in the duration from 1992 to 1995, the isolates of C. indologenes were least susceptible to trimethoprim-sulfamethoxazole (30.6 %) followed by 41.Our study had certain limitations.Firstly, it is a single centre study with very few isolates that does not represent the picture of infections in the other hospitals in our geographical location.Secondly, it is a retrospective study where all the data was extracted from the patient records which could lead to investigator bias or information bias.Thirdly, we could not perform broth microdilution (BMD) method of antibiotic susceptibility testing on these isolates as all isolates were recorded retrospectively.The above limitation also includes a lack of standardization in interpretation of breakpoints for susceptibility testing (both disc diameter and Epsilometeric-test) for Chryseobacterium, in addition to not performing broth microdilution (BMD).

CONCLUSION
To the best of our knowledge, this is the first study from northern India that reported clinical characteristics and an antimicrobial susceptibility profile of Chryseobacterium spp.With the advent of MALDI-TOF MS, early and accurate identification of these emerging microorganisms facilitates management of infection.Very limited data is available on the antimicrobial susceptibility pattern of Chryseobacterium spp., so there is a need to determine the susceptibility data of this microorganism worldwide to provide a uniform platform for performing antimicrobial susceptibility testing.Minocycline is a suitable antibiotic for Chryseobacterium bacteremia.The Microbiology Society is a membership charity and not-for-profit publisher.
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Comments: 1. Methodological rigour, reproducibility and availability of underlying data Additional information were added following the reviewers' recommendation 2. Presentation of results Revision were done and additional information added following the reviewers' recommendation 3. How the style and organization of the paper communicates and represents key findings As the mentioned in my 1st review, most of the key findings had been presented in concise manner 4. Literature analysis or discussion The authors had made the discussion more comprehensive 5. Any other relevant comments Some of the antibiotics names had their first character being capitalized, while others were not.This might need to be standardized: -line 76: "Tigecycline", "Colistin" -small "t" and "c" -line 83: "Macrolide" -small "m" -line 126: Cefoperazone-sulbactam -small "c" -line 261: "Piperacillin" -small "p" Same for line 160: "Gall bladder" -small "g"

Please rate the quality of the presentation and structure of the manuscript Good
To what extent are the conclusions supported by the data?Strongly support

Is there a potential financial or other conflict of interest between yourself and the author(s)? No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes 1 A native English speaker should review the manuscript and check all the grammatical errors.Although I have corrected many grammatical errors throughout the manuscript, further proofreading is required.
We have reviewed the manuscript for all grammatical errors and proofreading has been done by us.

2
Introduction-line 80: The name of genes must be written italics throughout the text.
The genes have been written in italics throughout the text.The necessary correction has been made and highlighted in the revised manuscript.

Response to reviewer 1
Serial No. Reviewers' comment Authors' response to reviewer line 120-"spp."should not be written italics "spp." has been corrected throughout the revised manuscript.Line 120-Please delete the phrase " antimicrobial susceptibility testing" The phrase has been deleted from the revised manuscript.line 123-Please delete "piperacillin-tazobactam" We have deleted the name of the antibiotic in the revised manuscript.line 126-Please clarify exactly that, based on the which criteria in the CLSI guideline, the AST results were interpreted via the disc diffusion method.
As there are no defined guidelines for reporting Antimicrobial Susceptibility Testing (AST) for Chryseobacteriumspp., we performed AST by Kirby-Bauer disc diffusion method on Müller Hinton agar using the antibiotic discs.line 128-the use The necessary correction has been made and highlighted in the revised manuscript.line 129-Please add a dot at the end of the sentence.
A dot has been added at the end of the sentence in the revised manuscript.results-line 136-It is better to write:...In our study, a total of 42 isolates (Chryseobacterium indologenes, n = 40 (%=?); and Chryseobacterium gleum, n = 2 (%=?) were identified from bloodstream infections during the course of five years study period.
The correction as expected by the reviewer has been made and highlighted in the revised manuscript.line 137-If possible, please provide information on MALDI-TOF MS results in the results section.

Results of MALDI-TOF-MS have been added and highlighted in the revised manuscript. line 139-critical
The necessary correction has been made and highlighted in the revised manuscript.line 140-anesthesia The necessary correction has been made and highlighted in the revised manuscript.line 140-emergency The necessary correction has been made and highlighted in the revised manuscript.

Response to reviewer 1
Serial No. Reviewers' comment Authors' response to reviewer line 143-Please replace "on" with "or" The "on" in the statement has been replaced by "or" in the revised manuscript.line 143-Please delete the phrase "clinical diagnosis" The phrase "clinical diagnosis" has been removed from the statement in the revised manuscript.line 150-151: Based on the recommendation for line 136, it is better to delete the sentence.
The statement in line number 136 has been deleted in the revised manuscript.line 152-153-In this study, only isolates recovered from the bloodstream infections were included in the study and there was no data about the prevalence of bacteria in another clinical specimen.So, in my opinion, it is better to move this sentence (line 152-153) to the discussion section and discuss this issue based on the results of previous studies.
The lines 152-153 have been moved to the discussion section and the issue has been discussed based on previous studies.line 155-157: In this section, only 19 cases from 30 cases with co-infections were mentioned with exact name of species.Please mention exact number / percentage for each species.
All the 30 species of the microorganisms causing co-infection along with Chryseobacteriumspp. have been added and highlighted in the revised manuscript.Please provide information on the prevalence of these 42 cases of Chryseobacterium infections by year during this 5-year study period in the form of a graph.
Figure 1 is the graph that has been added to provide information on the prevalence of these 42 cases of Chryseobacteriumbacteremia during the 5 -years of study period.line 158-Please rewrite the sentence:..." Treatment of all patients with bloodstream infections was empirically started on broad-spectrum antibiotics." The statement has been rewritten and highlighted in the revised manuscript.line 159-160: Please rewrite the sentence:..."The most common antibiotic prescribed in inpatient wards was meropenem-cilastin in...The statement has been rewritten and highlighted in the revised manuscript.line 164-cefoperazone (lowercase letter) The necessary correction has been made and highlighted in the revised manuscript.

Response to reviewer 1
Serial No. Reviewers' comment Authors' response to reviewer line 167-According to figure 1, you did not report any isolate with an intermediate pattern to antibiotics.Please mention this in the results section.
Figure 1 (previously) has been converted to Table 2 where the antibiotic susceptibility of all the isolates has been represented.None of the isolates reported intermediate sensitivity.The changed pattern of representation has also been mentioned in the results section.line 168-were The necessary correction has been made and highlighted in the revised manuscript.line 170-mortality rate The necessary correction has been made and highlighted in the revised manuscript.line 170-171-Rewrite the sentence so that the word "Table 2" is placed in parentheses at the end of the sentence.
Table 2 (previously) has been converted to Table 1 and added to the end of the sentence and highlighted in the revised manuscript.line 170-Please delete the number "54.76%" in the parenthesis.
The number "54.76%" in the parenthesis has been deleted in the revised manuscript.results-line 176-Please state the p-value for the risk factors or refer to the table.
The p-values for the risk factors have been added and highlighted in the revised manuscript.line 176-It is better to write "duration of hospitalization" instead of "length of hospital stay" The "length of hospital stay" has been replaced by "duration of hospitalization" and highlighted in the revised manuscript.line 182-Bacteremia Should not be written in italics.The necessary correction has been made and highlighted in the revised manuscript.line 182-..."Due to handful of patients..."This sentence is ambiguous.Please clarify the exact number and the antibiotic regimen of patients who survived.
The sentence has been clarified and the exact number and the antibiotic regimen of the patients who survived has also been clarified.Discussion-line 197-Please add more similar studies from other regions of the world and discuss your results and others.
More similar studies from other regions of the world have been added to discuss our results.

Response to reviewer 1 Serial No.
Reviewers' comment Authors' response to reviewer line 199-Please delete the word "our".
The word has been deleted in the revised manuscript.line 199-it is better to write..."were hospitalized in the critical care unit" instead of " came from critical care unit" The statement "came from critical care unit" has been replaced with the statement "were hospitalized in the critical care unit" in the revised manuscript.line 209-Please add more similar studies from other regions of the world and discuss your results and others.
More similar studies from other regions of the world have been added to the discussion of the results in the revised manuscript.line 210-"was" should not be written in italics The necessary correction has been made and highlighted in the revised manuscript.line 213-Please add more similar studies from other regions of the world and discuss your results and others.
More similar studies from other regions of the world have been added to the discussion of the results in the revised manuscript.line 219-Please add space between "to" and "minocycline" A space has been added between "to" and "minocycline".line 223-cefoperazone The necessary correction has been made and highlighted in the revised manuscript.line 225-it should be written AST instead of antibiotic susceptibility testing.
The necessary correction has been made and highlighted in the revised manuscript.line 226-Please rewrite the sentence "isolates by broth microdilution...." The sentence has been rewritten and highlighted in the revised manuscript.lines 229, 233, 235, 249: it should be written "susceptible" instead of "sensitive".
The term sensitive has been replaced with susceptible throughout the revised manuscript.line 224-Chryseobacteriumshould be written in italics.Chryseobacteriumhas been written in italics throughout the revised manuscript.Please update the references.Out of 21 references, only 4 were from 2019 and after.
The references have been updated.More references from 2019 and after have been added to the revised manuscript.

Response to reviewer 1 Serial No.
Reviewers' comment Authors' response to reviewer The taxonomic name of the species must be written in italics.Please correct this in the reference section.
The taxonomic name of the species has been written in italics.The taxonomic name of the species has also been corrected in the reference section.It is better to delete table 1

Response to reviewer 2 1
Topic: I suggest it should be modified as the study did not not describe the characteristics of Chryseobacterium species but rather cases where (associated) the pathogen was isolated.
The title of the manuscript has been changed to "Characteristics of Chryseobacteriumbacteremia, associated risk factors and their antibiotic susceptibility pattern at a university hospital: a descriptive, retrospective study" as more description of the characteristics of Chryseobacterium spp was not represented in the study.-under the section "Data Collection": Since this study collected data retrospectively over 5 years period, suggest to state the sources where the laboratory data and clinical information were retrieved retrospectively from, such as from Laboratory Information System (LIS), written worksheet, patient's medical records, or any others, etc.
-under the section "Antimicrobial Susceptibility Testing": as we understand that at this point, for Chryseobacteriumspecies, there is no interpretation breakpoints for both disc diffusion zone diameter and minimum inhibitory concentration (MIC), neither in CLSI or EUCAST guideline, which are the two most commonly used guideline in clinical microbiology laboratories worldwide.It is likely that various laboratories are interpreting the disc diffusion or MIC values using some kinds of presumptive interpretation, such as referring to the interpretation breakpoints of other organisms in similar categories.For the antibiotic susceptibility tests results reported in this retrospective study, how the disc inhibition zone diameter and/ or MIC value were interpreted in the authors' laboratory, means how the zone diameter/ MIC values obtained being concluded as "Sensitive" or "Resistant", were not being described.As in the "conclusion" section, line 258-259, the authors gave recommendation on the suitable antibiotic option to treat infection caused by this organism, based on the sensitivity test finding that 100% of Chryseobacteriumisolates in the authors' institution are sensitive to minocycline, hence it is important to describe the methodology of how the authors' laboratory interpret minocycline MIC or zone of inhibition as "sensitive".
-under the section "Data Collection": Since this study collected data retrospectively over 5 years period, the source of laboratory data has been mentioned to be derived from the laboratory registers and the clinical data was derived from the electronic hospital information system and patient files which has been added to the revised manuscript.
-under the section "Antimicrobial Susceptibility Testing": as we understand that at this point, for Chryseobacteriumspecies, there is no interpretation breakpoints for both disc diffusion zone diameter and minimum inhibitory concentration (MIC), neither in CLSI or EUCAST guideline, which are the two most commonly used guideline in clinical microbiology laboratories worldwide.The disc inhibition zone diameter determined by the Kirby-Bauer disc diffusion method and/ or MIC value determined by using the Epsilometeric -tests were interpreted in the laboratory using the susceptibility break points following the recommendation by the Clinical and Laboratory Standards Institute for non-Enterobacteriaceae and Pseudomonas aeruginosa.Chryseobacteriumspp. bacteremia): some of the abbreviations were not being listed.Row 9 (GIT) and row 10 (CVS), if these are referred to as "gastrointestinal tract" and "cardiovascular system", these are the human body's anatomical region or functional system, these are not underlying medical illness which predisposing patients to infections.Suggest specifically outline the types of gastrointestinal pathology and cardiovascular system disease that the patients are having.
-Figure 1 (Antimicrobial susceptibility profile of Chryseobacteriumspp.): as mentioned in the methodology section earlier, the authors described that both KB disc diffusion and VITEK 2 AST were being used to performed the antibiotic susceptibility on the Chryseobacteriumspp.Suggest to present the MIC values done by VITEK AST card.As for this scenario where there is no interpretation breakpoints in reference guideline, and if the laboratories are doing presumptive interpretation on the susceptibility test results, interpretation based on MIC values will be more accurate compared to disc diffusion.
-For the report on antibiotic therapy received by the patients with Chryseobacteriumspp., instead of mentioning only the types of antibiotic agents being administered, the presentation could be made more complete by also mentioning the mode of drug administration (such as oral or intravenous) and the duration of antibiotics, if not the dosage regime (understand that this manuscript may not having collaboration with Infectious Disease physicians or clinical pharmacist).As the authors also commented about the patients' outcome and gave recommendation on the suitable antibiotic option for treating infection caused by this organism, hence the prospective readers will be interested to know the mode of administration and duration of antibiotics given, especially in cases with successful treatment.
-The Table 1 and Table 2 (denoted before revision) has been removed and the diseases related to the two organ systems have been included in the underlying comorbidities section of Table 1 (after revision).
The type of gastrointestinal pathologies and cardiovascular diseases has been highlighted in the results section in the revised document.
-Figure 1 has been changed to a tabular form as We have tried to present every aspect of the study in a concise manner.As expected by the reviewers we have elaborated on some necessary points.

4
Literature analysis or discussion: If the authors are clinical microbiologists and/ or microbiology laboratory personnel, the limitations also include lack of standardization in interpretation breakpoints for susceptibility testing (both disc diameter and MIC) for Chryseobacterium, in addition to not performing broth microdilution (BMD).
The limitations including the lack of standardization in interpretation breakpoints for susceptibility testing (both disc diameter and MIC) for Chryseobacterium, in addition to not performing broth microdilution (BMD) have been added to the limitations in the section of discussion.
Comments: Dear Sir/ Madam, Please find my humble comments and suggestions below.1. Methodological rigour, reproducibility and availability of underlying data: -under the section "Data Collection": Since this study collected data retrospectively over 5 years period, suggest to state the sources where the laboratory data and clinical information were retrieved retrospectively from, such as from Laboratory Information System (LIS), written worksheet, patient's medical records, or any others, etc. -under the section "Antimicrobial Susceptibility Testing": as we understand that at this point, for Chryseobacterium species, there is no interpretation breakpoints for both disc diffusion zone diameter and minimum inhibitory concentration (MIC), neither in CLSI or EUCAST guideline, which are the two most commonly used guideline in clinical microbiology laboratories worldwide.It is likely that various laboratories are interpreting the disc diffusion or MIC values using some kinds of presumptive interpretation, such as referring to the interpretation breakpoints of other organisms in similar categories.For the antibiotic susceptibility tests results reported in this retrospective study, how the disc inhibition zone diameter and/ or MIC value were interpreted in the authors' laboratory, means how the zone diameter/ MIC values obtained being concluded as "Sensitive" or "Resistant", were not being described.As in the "conclusion" section, line 258-259, the authors gave recommendation on the suitable antibiotic option to treat infection caused by this organism, based on the sensitivity test finding that 100% of Chryseobacterium isolates in the authors' institution are sensitive to minocycline, hence it is important to describe the methodology of how the authors' laboratory interpret minocycline MIC or zone of inhibition as "sensitive".2. Presentation of results: -Table 1 (Predisposing risk factors in patients with Chryseobacterium spp.bacteremia): some of the abbreviations were not being listed.Row 9 (GIT) and row 10 (CVS), if these are referred to as "gastrointestinal tract" and "cardiovascular system", these are the human body's anatomical region or functional system, these are not underlying medical illness which predisposing patients to infections.Suggest specifically outline the types of gastrointestinal pathology and cardiovascular system disease that the patients are having.-Figure 1 (Antimicrobial susceptibility profile of Chryseobacterium spp.): as mentioned in the methodology section earlier, the authors described that both KB disc diffusion and VITEK 2 AST were being used to performed the antibiotic susceptibility on the Chryseobacterium spp.Suggest to present the MIC values done by VITEK AST card.As for this scenario where there is no interpretation breakpoints in reference guideline, and if the laboratories are doing presumptive interpretation on the susceptibility test results, interpretation based on MIC values will be more accurate compared to disc diffusion.-For the report on antibiotic therapy received by the patients with Chryseobacterium spp., instead of mentioning only the types of antibiotic agents being administered, the presentation could be made more complete by also mentioning the mode of drug administration (such as oral or intravenous) and the duration of antibiotics, if not the dosage regime (understand that this manuscript may not having collaboration with Infectious Disease physicians or clinical pharmacist).As the authors also commented about the patients' outcome and gave recommendation on the suitable antibiotic option for treating infection caused by this organism, hence the prospective readers will be interested to know the mode of administration and duration of antibiotics given, especially in cases with successful treatment.3. How the style and organization of the paper communicates and represents key findings: Most of the important key findings were outlined in concise manner.4. Literature analysis or discussion: If the authors are clinical microbiologists and/ or microbiology laboratory personnel, the limitations also includes lack of standardization in interpretation breakpoints for susceptibility testing (both disc diameter and MIC) for Chryseobacterium, in addition to not performing broth microdilution (BMD).

Fig. 1 .
Fig. 1.The prevalence of the 42 cases of Chryseobacterium bacteremia by year during the 5 year study period (N=42).

Table 1 .
Comparison of demographic and clinical characteristics among the Chryseobacterium bacteremia patients who died or survived (n=42)

Table 2 .
Antimicrobial susceptibility profile of Chryseobacterium spp.isolated from bloodstream infections of patients included in our study cohort (n=42) According to this study and previously published studies minocycline, trimethoprim-sulfamethoxazole and doxycycline were the most effective antibiotics in treatment of Chryseobacterium bacteremia.Among the patients who survived in this study, only five (5/19, 26.32 %) patients received appropriate antibiotics whereas seven (7/23, 30.43 %) patients who died had received appropriate antibiotics.On comparison of drug administration among the two groups, more adequate antibiotic administration was observed in the patients who died which could be attributed to the delayed administration of appropriate antibiotic therapy associated with adverse outcome as observed in a study by Falcone et al.[27].
and transfer the information from table 1 to table 2. Please add the p-value for these risk factors and add it to table 2. Please add CVS and GIT in the abbreviations section and write "history of " instead of H/O.Finally, please remove "table 1" and correct the number of table 2 throughout the manuscript.Agreeing with the reviewers' comments, table 1 has been deleted and the p-value for risk factors has been added to the table 2 which is now added to revised manuscript.CVS and GIT has been expanded and written in the table 1 (new).H/O has also been written as "history of ".The Table 1 (previously) has been removed and the number of Table 2 has been changed throughout the manuscript.In my opinion, the graph could not represent exact information about the AST.Please delete figure 1 and present the data of AST as a table and mention the number/% of isolates susceptible, intermediate or resistant to each antibiotic.

Table 1 (
Predisposing risk factors in patients with

Table 2
Topic: I suggest it should be modified as the study did not not describe the characteristics of Chryseobacterium species but rather cases where (associated) the pathogen was isolated.Lines 112-113: Do you mean to say positive blood culture from samples collected within 48 hours of admission?Authors may recast statement Lines 117-126: Since no interpretation guidelines for antimicrobial susceptibility currently exist for the pathogen, what was the basis used to categorize isolates as resistant or susceptible to each antimicrobial agent tested.Lines 182-184: Could the high rate of meropenem resistance you reported be attributed to the use of the drug as the empirical choice for treatment in the hospital The current study deals with the "Clinical and microbiological characteristics of Chryseobacterium species isolates from bloodstream infections from a university hospital: a descriptive, retrospective study".Isolates identified and characterized by conventional and MALDI-TOF MS methods and antibiotic susceptibility pattern.The results of this study are interesting and of great importance in respect to the clinical microbiology.However I have several comments which need addressing: 1-A native English speaker should review the manuscript and check all the grammatical errors.Although I have corrected many grammatical errors throughout the manuscript, further proofreading is required.2-Introduction-line80:Thename of genes must be written italics throughout the text.3-line90-itshouldbewritten..."and" 4-materials and methods: line 97-Please clarify that did you find any isolate with the taxonomic name Flavobacterium (the previous name of this genus) in the study.Based on the recommendation for line 136, it it better to delete the sentence.23-line152-153-Inthisstudy,onlyisolatesrecoveredfromthebloodstreaminfectionswereincludedinthestudyand there was no data about the prevalence of bacteria in another clinical specimen.So, in my opinion, it is better to move this sentence (line 152-153) to the discussion section and discuss this issue based on the results of previous studies.24-line155-157:Inthissection,only19casesfrom30caseswithco-infectionswerementionedwithexact name of species.Please mention exact number / percentage for each species.25-results:Pleaseprovideinformationon the prevalence of these 42 cases of Chryseobacterium infections by year during this 5-year study period in the form of a graph.26-line158-Pleaserewrite the sentence:..." Treatment of all patients with bloodstream infections was empirically started on broad-spectrum antibiotics."27-line159-160:Pleaserewritethesentence:..."The most common antibiotic prescribed in inpatient wards was meropenem-cilastine in... 28-line 164-cefoperazone (lowercase letter) 29-line 167-According to figure1, you did not report any isolate with an intermediate pattern to antibiotics.Please mention this in the results section.30-line168-were31-line170-mortalityrate32-line170-171-Rewrite the sentence so that the word "Table2" is placed in parentheses at the end of the sentence.33-line170-Pleasedeletethenumber"54.76%" in the parenthesis.34-results-line176-Pleasestatethep-valuefortheriskfactorsorrefertothe table.35-line176-It is better to write "duration of hospitalization" instead of "length of hospital stay" 36-line 182-Bacteremia Should not be written in italics.37-line182-..."Due to handful of patients..."This sentence is ambiguous.Please clarify the exact number and the antibiotic regimen of patients who survived.38-Discussion-line197-Pleaseaddmoresimilarstudiesfromotherregions of the world and discuss your results and others.39-line199-Pleasedelete the word "our".40-line199-it is better to write..."were hospitalized in the critical care unit" instead of " came from critical care unit" 41-line 209-Please add more similar studies from other regions of the world and discuss your results and others.42-line210-"was"shouldnotbewritten in italics 43-line 213-Please add more similar studies from other regions of the world and discuss your results and others.44-line219-Pleaseaddspacebetween"to"and"minocycline"45-line223-cefoperazone46-line225-itshould be written AST instead of antibiotic susceptibility testing 47-line 226-Please rewrite the sentence "isolates by broth microdilution...." 48-lines 229, 233, 235, 249: it should be written "susceptible" instead of "sensitive" 49-line 224-Chryseobacterium should be written in italics.50-Pleaseupdate the references.Out of 21 references, only 4 were from 2019 and after.51-Thetaxonomicname of the species must be written in italics.Please correct this in the reference section.52-It is better to delete table 1 and transfer the information from table 1 to table 2. Please add the p-value for these risk factors and add it to table 2. Please add CVS and GIT in the abbreviations section and write "history of " instead of H/O.Finally, please remove "table 1" and correct the number of table 2 throughout the manuscript.53-Inmyopinion, the graph could not represent exact information about the AST.Please delete figure1and present the data of AST as a table and mention the number/% of isolates susceptible, intermediate or resistant to each antibiotic.54-Ifinformation is available, please add the number/ % of survived and dead patients with co-infection in the table 2. 55-Table2-survived(n=19) 56-Table2-Of 19 surviving patients with bacteremia, 73.7% did not receive appropriate antibiotics.Of 23 dead patients, in comparison with surviving patients, a higher number of them received suitable antibiotics.According to the literature review, please discuss possible reasons for surviving patients.Please rate the manuscript for methodological rigour GoodPlease rate the quality of the presentation and structure of the manuscript GoodTo what extent are the conclusions supported by the data?Strongly supportDo you have any concerns of possible image manipulation, plagiarism or any other unethical practices?NoIs there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes ://doi.org/10.1099/acmi.0.000594.v1.2 © 2023 The Authors.This is an open-access article report distributed under the terms of the Creative Commons License.
https://doi.org/10.1099/acmi.0.000594.v1.5 © 2023 Anonymous.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?NoIs there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes -Line 120-Please delete the phrase " antimicrobial susceptibility testing" 11-line 123-Please delete "piperacillin-tazobactam" 12-line 126-Please clarify exactly that, based on the which criteria in the CLSI guideline, the AST results were interpreted via the disc diffusion method.13-line 128-the use 14-line 129-Please add a dot at the end of the sentence.15-results-line 136-It is better to write:...In our study, a total of 42 isolates (Chryseobacterium indologenes, n = 40 (%=?); and Chryseobacterium gleum, n = 2 (%=?) were identified from bloodstream infections during the course of five years study period.16-line 137-If possible, please provide information on MALDI-TOF MS results in the results section.17-line 139-critical 18-line 140-anesthesia 19-line 140-emergency 20-line 143-Please replace "on" with "or" 21-line 143-Please delete the phrase "clinical diagnosis" 22-line 150-151: https://doi.org/10.1099/acmi.0.000594.v1.1 © 2023 The Authors.This is an open-access article report distributed under the terms of the Creative Commons License.iThenticate report https